IGF-I fusion polypeptides and therapeutic uses thereof

ABSTRACT

A fusion protein comprising at least one IGF1 variant component and a fusion component (F), and, optionally, a signal sequence, exhibiting improved stability relative to the native IGF1 or IGF2 polypeptide. The fusion component (F) may be a multimerizing component, a targeting ligand, or another active or therapeutic compound. IGF1 variants were shown to have improved ability to induce skeletal muscle hypertrophy relative to native IGF1.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 USC § 119(e) of U.S.Provisionals 60/642,229 filed 7 Jan. 2005 and 60/656,583 filed 25 Feb.2005, which applications are herein specifically incorporated byreference in their entirety.

BACKGROUND

1. Field of the Invention

This invention relates to insulin-like growth factor I (IGFI) andinsulin-like growth factor 2 (IGF2) polypeptides, methods of producingsuch polypeptides, and therapeutic methods for administering suchpolypeptides.

2. Description of Related Art

The insulin-like growth factors (IGFs) constitute a family of proteinshaving insulin-like and growth stimulating properties. The IGFs showclose structural homology with proinsulin and elicit similar biologicaleffects. Human IGFI (also known as somatomedin C) is a 70 aa basicpeptide (pl 8.4) having the protein and DNA sequences shown in SEQ IDNOs:1-2, and has a 43% homology with proinsulin (Rinderknecht et al.(1978) J. Biol. Chem. 253:2769-2776). Human IGF2 is a 67 amino acidbasic peptide having the protein and DNA sequences shown in SEQ IDNOs:3-4. Specific binding proteins of high molecular weight having veryhigh binding capacity for IGF1 and IGF2 act as carrier proteins or asmodulators of IGF1 functions (Holly et al. (1989) J. Endocrinol.122:611-618).

IGFI and IGF2 and variants thereof have been used to treat humanssuffering from growth hormone deficiencies, tissue wasting includingburns, skeletal trauma, infection, cancer, cystic fibrosis, Duchennemuscular dystrophy, Becker dystrophy, autosomal recessive dystrophy,polymyositis, as well as myopathies and AIDS (U.S. Pat. No. 5,622,932).

BRIEF SUMMARY OF THE INVENTION

In the broadest embodiment, the present invention comprises compositionsand methods providing IGF1 and IGF2 variant molecules to a subject inneed thereof. More specifically, the present invention provides fusionpolypeptides comprising a therapeutic IGF1 or IGF2 variants or analogsthereof fused to a fusion component (F). The fusion polypeptides of theinvention are capable of remaining therapeutically active and availablefor a longer period of time than the naturally occurring molecule andresist inactivation by an IGF binding protein. The fusion polypeptidesof the invention can also be used in a variety of in vitro and in vivodiagnostic and prognostic assays.

In a first aspect, the invention features an IGF1 fusion polypeptide,comprising (a) at least one IGF1 variant component, (b) a fusioncomponent (F), and optionally, (c) a signal sequence, wherein the IGFvariant component is the human IGF1 protein of SEQ ID NO:1 comprising(i) a modification of the C-terminus selected from the group consistingof deletion of 3 to 6 amino acids, e.g., 68-70(Δ68-70), Δ67-70, Δ66-70,Δ65-70, deletion of Lys68(Δ68), substitution of amion acid 68 withanother amino acid, deletion of amino acids 65-70(Δ65-70), deletion ofLys65(Δ65), and substitution of amino acid 65 with another amino acid;(ii) a modification of the N-terminus selected from the group consistingof deletion of amino acids 1-3 (Δ1-3) and substitution of Glu3 with adifferent amino acid, and/or (iii) a modification at Arg36 and/or Arg37selected from the group consisting of deletion of Arg36 (Δ36), deletionof Arg 37 (Δ37), substitution of Arg36 with a different amino acid,e.g., Arg36Ala, and substitution of Arg37 with a different amino acid,e.g., Arg37Ala. In a specific embodiment, the IGF1 fusion protein has adeletion of amino acids 1-3 and Arg37 (2D-IGF1-Fc)(Δ1-3, ΔArg37). Inanother specific embodiment, the IGF1 fusion protein has a deletion ofamino acids 1-3, Arg37 and amino acids 68-70 (3D-IGF1-Fc)(Δ1-3, ΔArg37,Δ68-70).

In a second aspect, the invention features an IGF2 fusion polypeptide,comprising (a) at least one IGF2 variant component, (b) a fusioncomponent (F), and optionally, (c) a signal sequence, wherein the IGFvariant component is the human IGF2 protein of SEQ ID NO:3 comprising(i) a modification of the C-terminus selected from the group consistingof deletion of 3 amino acids, e.g., 65-67(Δ65-67), deletion ofLys65(Δ65), and substitution of amino acid 65 with another amino acid;(ii) a modification of the N-terminus selected from the group consistingof deletion of amino acids 1-6 (Δ1-6) and substitution of Glu6 with adifferent amino acid, and/or (iii) a modification at Arg37 and/or Arg38selected from the group consisting of deletion of Arg37 (Δ37), deletionof Arg 38 (Δ38), substitution of Arg37 with a different amino acid,e.g., Arg37Ala, and substitution of Arg38 with a different amino acid,e.g., Arg38Ala.

The fusion component (F) is any component that enhances thefunctionality of the fusion polypeptide. Thus, for example, an F mayenhance the biological activity of the fusion polypeptide, aid in itsproduction and/or recovery, or enhance a pharmacological property or thepharmacokinetic profile of the fusion polypeptide by, for example,enhancing its serum half-life, tissue penetrability, lack ofimmungenicity, or stability. In a preferred embodiment, the fusioncomponent allows the IGF variant component to evade serum bindingproteins which may sequester IGF into a less biologically activecompartment.

In preferred embodiments, F is a multimerizing component from the groupconsisting of (i) an amino acid sequence between 1 to about 500 aminoacids in length, optionally comprising at least one cysteine residue,(ii) a leucine zipper, (iii) a helix loop motif, (iv) a coil-coil motif,and (v) an immunoglobulin domain. In some embodiments, the fusioncomponent comprises an immunoglobulin-derived domain from, for example,human IgG, IgM or IgA. In specific embodiments, theimmunoglobulin-derived domain is selected from the group consisting ofthe Fc domain and the heavy chain of IgG. The Fc domain of IgG may beselected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as anyallotype within each isotype group.

In a specific embodiment, the invention features an IGF1 fusionpolypeptide, comprising (i) an IGF1 variant component comprising thehuman IGF1 protein of SEQ ID NO:1 comprising deletion of amino acids 1-3(Δ1-3 or delGPE), deletion of Arg36 (Δ36), and a deletion of 3-6 aminoacids at the C-terminus(Δ68-70), (ii) an Fc domain of an IgG, andoptionally, (iii) a signal sequence.

In another specific embodiment, the invention features an IGF2 fusionpolypeptide, comprising (i) an IGF2 variant component comprising thehuman IGF2 protein of SEQ ID NO:3 comprising deletion of amino acids 1-6(Δ1-6 or delAYRPSE), deletion of Arg37 (Δ37), and a deletion of 3 aminoacids at the C-terminus (Δ65-67), (ii) an Fc domain of an IgG, andoptionally, (iii) a signal sequence.

In other embodiments, the fusion component (F) is a targeting ligand, orderivative or fragment thereof, capable of binding specifically to apre-selected cell surface protein, and thereby delivering said IGF1 orIGF2 to a target cell, e.g. a muscle cell. In specific embodiments, thetargeting component is MuSK ligand, or a fragment of a MuSK ligandcapable of binding the MuSK receptor. In specific embodiments, theMuSK-specific ligand is agrin or a fragment or derivative thereofcapable of binding MuSK, or an anti-MuSK antibody or fragment orderivative thereof, including, for example, an scFv. In other specificembodiments, the muscle-targeting ligand of the muscle-targeting fusionpolypeptide comprises three or more muscle cadherin (M-cadherin)extracellular cadherin domains, or derivatives or fragments thereof,capable of binding specifically to a muscle cells or other cells thatexpress homophilic muscle cadherins. In one specific embodiment, themuscle-targeting ligand consists essentially of the first three (3) orfour (4) N-terminal extracellular domains of M-cadherin.

In other embodiments, the fusion component (F) of the invention isanother active compound, which may be any agent that is desirable todeliver to a pre-selected site for therapeutic purposes. In specificembodiments, the active or therapeutic agent is a ligand for a secondcell surface receptor, and is capable of binding and activating a secondreceptor. In other embodiments, the active or therapeutic agent is anagent capable of blocking the activity of another agent that is activeon the target cell. In a specific embodiment, the active or therapeuticagent is selected from the group consisting of IL-15, myotrophin,urocortin, urocortin II, a natural or mutant IGF1 or IGF2, insulin, thepro domain of myostatin, hGH, proliferin, follistatin, FSTL1, and FLRG,and a biologically active fragments thereof.

The polypeptide or fusion polypeptide of the invention may furtheroptionally encode a signal sequence (SS) component. When a SS is part ofthe polypeptide, any SS known to the art may be used, includingsynthetic or natural sequences from any source, for example, from asecreted or membrane bound protein. Generally, a signal sequence isplaced at the beginning or amino-terminus of the fusion polypeptide ofthe invention.

The components of the fusion polypeptides of the invention may beconnected directly to each other or connected via one or more spacersequences. In one preferred embodiment, the components are fuseddirectly to each other. In another preferred embodiment, the componentsare connected with a spacer of 1-200 amino acids. Any spacer known tothe art may be used to connect the polypeptide components. A spacersequence may also include a sequence used to enhance expression of thefusion polypeptide, provide restriction sites, allow component domainsto form optimal tertiary and quaternary structures and/or to enhance theinteraction of a component with its receptor. In one embodiment, thefusion polypeptide of the invention comprises one or more peptidesequences between one or more components which is (are) between 1-25amino acids.

The components of the fusion polypeptide of the invention may bearranged in a variety of configurations and may comprise more than oneIGF variant polypeptide, for example, IGF-F; IGF-IGF-F; IGF-F-IGF;F-IGF; F-IGF-IGF, etc. However, when F is an Fc, the Fc must be on the Cterminus of the fusion polypeptide. Similarly, in fusions comprising asecond active component, such as human growth hormone (hGH), theconfiguration must be IGF variant-hGH.

In a second aspect, the invention features a nucleic acid encoding afusion polypeptide of the invention.

In a third aspect, the invention features a vector comprising a nucleicacid molecule of the invention. In further fourth and fifth aspects, theinvention encompasses vectors comprising the nucleic acid molecules ofthe invention, including expression vectors comprising the nucleic acidmolecules operatively linked to an expression control sequence, andhost-vector systems for the production of a fusion polypeptide whichcomprise the expression vector, in a suitable host cell; host-vectorsystems wherein the suitable host cell is, without limitation, abacterial, yeast, insect, or mammalian cell. Examples of suitable cellsinclude E. coli, B. subtilis, BHK, COS and CHO cells. Additionallyencompassed are fusion polypeptides of the invention modified byacetylation or pegylation. Methods for acetylating or pegylating aprotein are well known in the art.

In a related sixth aspect, the invention features a method of producinga fusion polypeptide of the invention, comprising culturing a host celltransfected with a vector comprising a nucleic acid molecule of theinvention, under conditions suitable for expression of the protein fromthe host cell, and recovering the polypeptide so produced.

In a seventh aspect, the invention features therapeutic methods for thetreatment of a disease or condition, comprising administering atherapeutically effective amount of the IGF fusion protein of theinvention to a subject in need thereof, or a subject at risk fordevelopment of that disease or condition. When the disease or conditionis a muscle condition, such as atrophy, the therapeutic method of theinvention comprises administering a therapeutically effective amount ofan IGF fusion protein of the invention to a subject in need thereof,wherein the muscle-related disease or condition is ameliorated orinhibited. The muscle-related condition or disorder treated by thefusion polypeptides of the invention may arise from a number of sources,including for example: denervation; degenerative, metabolic orinflammatory neuropathy; infantile and juvenile spinal muscularatrophies; autoimmune motor neuropathy; from chronic disease, includingcachexia resulting from cancer, AIDS, fasting or rhabdomyolysis; andfrom muscular dystrophy syndromes such as Duchenne. The therapeuticmethod of the invention are useful to treat any condition which isresults from an IGF deficiency or which may be improved by increased IGFlevels, including dwarfism and heart disease, for example, improvedheart tissue survival following myocardial infarction.

Accordingly, in an eighth aspect, the invention features pharmaceuticalcompositions comprising a fusion protein of the invention with apharmaceutically acceptable carrier. Such pharmaceutical compositionsmay comprise the fusion proteins or nucleic acids which encode them,together with a pharmaceutically acceptable carrier.

Other objects and advantages will become apparent from a review of theensuing detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the serum concentration of IGF1 variants2D-IGF1-Fc (□) or 3D-IGF1-Fc (▪) in CD-1 mice at 0, 24, 48, 72, 96, 120,144 and 168 hrs (n=5 per group)(Mean±SEM).

DETAILED DESCRIPTION

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, references to “a method”include one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference.

General Description

The invention encompasses fusion polypeptides and nucleic acids thatencode them which comprise one or more IGF variant components and afusion component (F), which may comprise a multimerizing component, atargeting component, and/or one or more additional active or therapeuticagent(s).

Definitions

“Biologically active” fragments or derivatives of a component of thefusion polypeptides of the invention encompass any naturally occurringmolecule, or mutant or derivative thereof capable of achieving thedesired effect at the target site. For example, described herein arevariants of IGF1, which have improved properties of activity andstability. The invention envisions the use of a mutant or derivative ofthe IGF1 molecules described herein which are capable of binding theIGF1 receptor. A “biologically active” fragment of derivative of anytargeting component is any portion or mutant thereof capable of bindingthe target cell. Thus, for example, when the targeting ligand is agrin,a biologically active fragment or derivative is any portion or mutant ofagrin capable of binding the MuSK receptor.

The terms “treatment”, “treating”, and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease, condition, or symptoms thereof, and/ormay be therapeutic in terms of a partial or complete cure for a diseaseor condition and/or adverse effect attributable to the disease orcondition. “Treatment” as used herein covers any treatment of a diseaseor condition of a mammal, particularly a human, and includes: (a)preventing the disease or condition from occurring in a subject whichmay be predisposed to the disease or condition but has not yet beendiagnosed as having it; (b) inhibiting the disease or condition, i.e.,arresting its development; or (c) relieving the disease or condition,i.e., causing regression of the disease or condition. The population ofsubjects treated by the method of the disease includes subject sufferingfrom the undesirable condition or disease, as well as subjects at riskfor development of the condition or disease.

By the term “therapeutically effective dose” is meant a dose thatproduces the desired effect for which it is administered. The exact dosewill depend on the purpose of the treatment, and will be ascertainableby one skilled in the art using known techniques (see, for example,Lloyd (1999) The Art, Science and Technology of PharmaceuticalCompounding).

As used herein, a “condition or disease” generally encompasses acondition of a mammalian host, particularly a human host, which isundesirable and/or injurious to the host. Thus, treating amuscle-related condition with a fusion polypeptide which specificallytargets skeletal muscle will encompass the treatment of a mammal, inparticular, a human, who has symptoms reflective of decreased targetmuscle receptor activation, or who is expected to have such decreasedlevels in response to a disease, condition or treatment regimen.Treating a muscle-related condition or disease encompasses the treatmentof a human subject wherein enhancing the activation of a target musclereceptor with the muscle specific fusion polypeptide of the inventionresults in amelioration of an undesirable symptom resulting from themuscle-related condition or disease. As used herein, a “muscle-relatedcondition” also includes a condition in which it is desirable to alter,either transiently, or long-term, activation of a particular targetmuscle receptor.

IGF1 or IGF2 Variant Component

The first component of the polypeptides of the invention is an IGF1 orIGF2 variant (“IGF variant”). In the case of IGF1, such variantscomprise mature human IGF1 (SEQ ID NO:1) having the followingmodifications: (i) a deletion of 3-6 amino acids at the C-terminus,e.g., Lys65 to Ala70; (ii) a modification at the N-terminus selectedfrom the group consisting of deletion of amino acids 1-3 andsubstitution of Glu3 with a different amino acid, such as a alanine,valine, histidine or arginine, e.g., Glu3Arg or Glu3Ala, and/or (iii) amodification at Arg36 and/or Arg37 selected from the group consisting ofdeletion of Arg36, deletion of Arg 37, substitution of Arg36 with adifferent amino acid, e.g., Arg36Ala, and substitution of Arg37 with adifferent amino acid, e.g., Arg37Ala.

In the case of IGF2, such variants comprise the human IGF2 protein ofSEQ ID NO:3 comprising (i) a modification of the C-terminus selectedfrom the group consisting of deletion of 3 amino acids, e.g.,65-67(Δ65-67), deletion of Lys65(Δ65), and substitution of amino acid 65with another amino acid; (ii) a modification of the N-terminus selectedfrom the group consisting of deletion of amino acids 1-6 (Δ1-6) andsubstitution of Glu6 with a different amino acid, such as alanine,valine, histidine or arginine, e.g. Glu6Arg or Glu6Ala; and/or (iii) amodification at Arg37 and/or Arg38 selected from the group consisting ofdeletion of Arg37 (Δ37), deletion of Arg 38 (Δ38), substitution of Arg37with a different amino acid, e.g., Arg37Ala, and substitution of Arg38with a different amino acid, e.g., Arg38Ala.

Such modifications prevent the cleavage of the fusion component from theIGF1 or IGF2 variant, thus enhancing its stability and half-life.

Targeting Ligand Component

In some embodiments, the fusion component of the fusion polypeptides ofthe invention is a targeting ligand. A targeting ligand is a molecule,e.g., a protein or fragment thereof that specifically binds with highaffinity to a target on a pre-selected cell, such as a surface proteinsuch as a receptor that is present to a greater degree on thepre-selected cell target than on any other body tissue. For example, asdescribed in U.S. Pat. Nos. 5,814,478 and 6,413,740, the MuSK receptoris highly specific to muscle. Accordingly, the cognate ligand agrin, aswell as MuSK binding portions thereof is an example of a targetingligand useful as a fusion component in the fusion polypeptides of thepresent invention. Another example of a targeting ligand is a group ofcadherin domains from a human cadherin. Accordingly, human cadherindomains from, for example, human muscle cadherin may be used in thetargeting fusion polypeptides of the invention to target muscle cells.The targeting ligand component of the fusion polypeptide of theinvention may include a naturally occurring or engineered ligand, or afragment thereof, capable of binding the pre-selected target cell.

In another embodiment of the invention, the targeting ligand componentof the targeting fusion polypeptides of the invention consists of atleast three, four or five muscle cadherin (M-cadherin) domains, orderivatives or fragments thereof, capable of binding specifically totarget cells that express homophilic cadherins. (Shimoyama et al. (1998)J. Biol. Chem. 273(16): 10011-10018; Shibata et al. (1997) J. Biol.Chem. 272(8):5236-5270). In preferred embodiments, the fusionpolypeptide of the invention comprises at least three cadherin domainsfrom the extracellular domain of human M-cadherin (or biologicallyactive fragments or derivatives thereof that are capable of bindinghomophilic M-cadherin), fused to the IGF1 variant component.

Further examples of targeting ligands also include, but are not limitedto, antibodies and portions thereof that bind a pre-selected cellssurface protein with high affinity. By “high affinity” is meant anequilibrium dissociation constant of at least 10⁻⁷ molar, as determinedby assay methods known in the art, for example, BiaCore analysis. In oneembodiment, the targeting ligand component of the targeting fusionpolypeptides of the invention may also comprise one or moreimmunoglobulin binding domains isolated from antibodies generatedagainst a selected tissue-specific surface protein or targettissue-specific receptor. The term “immunoglobulin or antibody” as usedherein refers to a mammalian, including human, polypeptide comprising aframework region from an immunoglobulin gene or fragments thereof thatspecifically binds and recognizes an antigen, which, in the case of thepresent invention, is a tissue-specific surface protein, a targettissue-specific receptor, or portion thereof. If the intended targetingfusion polypeptide will be used as a mammalian therapeutic,immunoglobulin binding regions should be derived from the correspondingmammalian immunoglobulins. If the targeting fusion polypeptide isintended for non-therapeutic use, such as for diagnostics and ELISAs,the immunoglobulin binding regions may be derived from either human ornon-human mammals, such as mice. The human immunoglobulin genes or genefragments include the kappa, lambda, alpha, gamma, delta, epsilon, andmu constant regions, as well as the myriad immunoglobulin variableregion genes. Light chains are classified as either kappa or lambda.Heavy chains are classified as gamma, mu, alpha, delta, or epsilon,which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, andIgE, respectively. Within each IgG class, there are different isotypes(e.g. IgG₁, IgG₂, etc.). Typically, the antigen-binding region of anantibody will be the most critical in determining specificity andaffinity of binding.

An exemplary immunoglobulin (antibody) structural unit of human IgG,comprises a tetramer. Each tetramer is composed of two identical pairsof polypeptide chains, each pair having one light chain (about 25 kD)and one heavy chain (about 50-70 kD). The N-terminus of each chaindefines a variable region of about 100-110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light chain”(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist as intact immunoglobulins, or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. For example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂, a dimer of Fab whichitself is a light chain joined to V_(H)-C_(H) by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region, thereby converting the F(ab)′₂ dimer intoan Fab′ monomer. The Fab′ monomer is essentially Fab with part of thehinge region. While various antibody fragments are defined in terms ofthe digestion of an intact antibody, one of skill will appreciate thatsuch fragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the terms immunoglobulin or antibody,as used herein, also includes antibody fragments either produced by themodification of whole antibodies, or those synthesized de novo usingrecombinant DNA methodologies (e.g., single chain Fv)(scFv)) or thoseidentified using phase display libraries (see, for example, McCaffertyet al. (1990) Nature 348:552-554). In addition, the fusion polypeptidesof the invention include the variable regions of the heavy (V_(H)) orthe light (V_(L)) chains of immunoglobulins, as well as tissue-specificsurface protein and target receptor-binding portions thereof. Methodsfor producing such variable regions are described in Reiter, et al.(1999) J. Mol. Biol. 290:685-698.

Methods for preparing antibodies are known to the art. See, for example,Kohler & Milstein (1975) Nature 256:495-497; Harlow & Lane (1988)Antibodies: a Laboratory Manual, Cold Spring Harbor Lab., Cold SpringHarbor, N.Y.). The genes encoding the heavy and light chains of anantibody of interest can be cloned from a cell, e.g., the genes encodinga monoclonal antibody can be cloned from a hybridoma and used to producea recombinant monoclonal antibody. Gene libraries encoding heavy andlight chains of monoclonal antibodies can also be made from hybridoma orplasma cells. Random combinations of the heavy and light chain geneproducts generate a large pool of antibodies with different antigenicspecificity. Techniques for the production of single chain antibodies orrecombinant antibodies (U.S. Pat. No. 4,946,778; U.S. Pat. No.4,816,567) can be adapted to produce antibodies used in the fusionpolypeptides and methods of the instant invention. Also, transgenicmice, or other organisms such as other mammals, may be used to expresshuman or humanized antibodies. Alternatively, phage display technologycan be used to identify antibodies, antibody fragments, such as variabledomains, and heteromeric Fab fragments that specifically bind toselected antigens.

Screening and selection of preferred immunoglobulins (antibodies) can beconducted by a variety of methods known to the art. Initial screeningfor the presence of monoclonal antibodies specific to a tissue-specificor target receptor may be conducted through the use of ELISA-basedmethods or phage display, for example. A secondary screen is preferablyconducted to identify and select a desired monoclonal antibody for usein construction of the tissue-specific fusion polypeptides of theinvention. Secondary screening may be conducted with any suitable methodknown to the art. One preferred method, termed “BiosensorModification-Assisted Profiling” (“BiaMAP”) is described in US patentpublication 2004/101920, allows rapid identification of hybridoma clonesproducing monoclonal antibodies with desired characteristics. Morespecifically, monoclonal antibodies are sorted into distinctepitope-related groups based on evaluation of antibody: antigeninteractions.

Active or Therapeutic Agent

In some embodiments, the fusion component (F) of the polypeptides of theinvention comprises a second active or therapeutic agent or mutant orderivative thereof, i.e. a molecule capable of having a desired effectwhen delivered to the pre-selected target site, e.g., cell or tissue.Active or therapeutic agents, include, but are not limited to, smallmolecules, hormones, growth factors, therapeutic biologics, activatingantibodies and portions thereof, and blocking antibodies and portionsthereof, that are capable of having a desirable effect upon delivery toa target cell or tissue.

In particular embodiments wherein the fusion polypeptide is directed atmuscle cells or tissue, the fusion polypeptide comprises a second activeor therapeutic agent that is active on muscle cells. Such agentsinclude, but are not limited to, insulin, IL-15, myotrophin, urocortin,urocortin II, human myostatin propeptide, a natural or mutant IGF1 orIGF2, hGH, proliferin, follistatin, FSTL1, and FLRG, or mutants,derivative, or fragments thereof having biologically activity. Inaddition, the active or therapeutic agent may comprise a blockingantibody or biologically active derivative thereof, which blocks, forexample, myostatin, activin receptor, BMP receptor 1, TNF receptor, IL-1receptor, ALK3 receptor and ALK4 receptor. Alternatively, the active ortherapeutic agent may comprise an activating antibody that activates,for example, the IFG1 receptor, B2adrenergic receptor or the IL-15receptor complex.

Multimerizing Component

In specific embodiments, the fusion component (F) of the fusionpolypeptides of the invention comprises a multimerizing component. Amultimerizing component includes any natural or synthetic sequencecapable of interacting with another multimerizing component to form ahigher order structure, e.g., a dimer, a trimer, etc. The multimerizingcomponent may be selected from the group consisting of an amino acidsequence between 1 to about 500 amino acids in length, a leucine zipper,a helix loop motif, and a coil-coil motif. When the multimerizingcomponent comprises an amino acid sequence between 1 to about 500 aminoacids in length, the sequence may contain one or more cysteine residuescapable of forming a disulfide bond with a corresponding cysteineresidue on another fusion polypeptide comprising a multimerizingcomponent with one or more cysteine residues. In some embodiments, themultimerizing component comprises an immunoglobulin-derived domain from,for example, human IgG, IgM or IgA, or comparable immunoglobulin domainsfrom other animals, including, but not limited to, mice. In specificembodiments, the immunoglobulin-derived domain may be selected from thegroup consisting of the constant region of IgG, the Fc domain of IgG, anFc-protein, and the heavy chain of IgG. The Fc domain of IgG may beselected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as anyallotype within each isotype group.

Component Spacers

The components of the targeting fusion polypeptides of the invention maybe connected directly to each other or be connected via spacers. Theterm “spacer” or “linker” means one or more molecules, e.g., nucleicacids or amino acids, or non-peptide moieties, such as polyethyleneglycol, which may be inserted between one or more component domains. Forexample, spacer sequences may be used to provide a restriction sitebetween components for ease of manipulation. A spacer may also beprovided to enhance expression of the fusion polypeptide from a hostcell, to decrease steric hindrance such that the component may assumeits optimal tertiary or quaternary structure and/or interactappropriately with its target molecule. For spacers and methods ofidentifying desirable spacers, see, for example, George et al. (2003)Protein Engineering 15:871-879, herein specifically incorporated byreference.

A spacer sequence may include one or more amino acids naturallyconnected to a receptor component, or may be an added sequence used toenhance expression of the fusion protein, provide specifically desiredsites of interest, allow component domains to form optimal tertiarystructures and/or to enhance the interaction of a component with itstarget molecule. In one embodiment, the spacer comprises one or morepeptide sequences between one or more components which is (are) between1-100 amino acids, preferably 1-25. In one specific embodiment, thespacer is a three amino acid sequence; more specifically, the threeamino acid sequence of Gly Pro Gly.

Nucleic Acid Construction and Expression

Individual components of the fusion polypeptides of the invention may beproduced from nucleic acids molecules using molecular biological methodsknown to the art. The nucleic acid of SEQ ID NO:2 or SEQ ID NO:4 withthe appropriate deletions or mutations may be used to prepare the IGF1or IGF2 variants described herein. Such nucleic acid molecules areinserted into a vector that is able to express the fusion polypeptideswhen introduced into an appropriate host cell. Appropriate host cellsinclude, but are not limited to, bacterial, yeast, insect, and mammaliancells. Any of the methods known to one skilled in the art for theinsertion of DNA fragments into a vector may be used to constructexpression vectors encoding the fusion polypeptides of the inventionunder control of transcriptional/translational control signals. Thesemethods may include in vitro recombinant DNA and synthetic techniquesand in vivo recombinations (See Sambrook et al. Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory; Current Protocols inMolecular Biology, Eds. Ausubel, et al., Greene Publ. Assoc.,Wiley-Interscience, NY).

Expression of the nucleic acid molecules of the invention may beregulated by a second nucleic acid sequence so that the molecule isexpressed in a host transformed with the recombinant DNA molecule. Forexample, expression of the nucleic acid molecules of the invention maybe controlled by any promoter/enhancer element known in the art.Promoters which may be used to control expression of the fusionpolypeptide molecules include, but are not limited to, the long terminalrepeat as described in Squinto et al. (1991) Cell 65:1-20; the SV40early promoter region, the CMV promoter, the M-MuLV 5′ terminal repeatthe promoter contained in the 3′ long terminal repeat of Rous sarcomavirus, the herpes thymidine kinase promoter, the regulatory sequences ofthe metallothionine gene; prokaryotic expression vectors such asthe-lactamase promoter, or the tac promoter (see also “Useful proteinsfrom recombinant bacteria” in Scientific American (1980) 242:74-94);promoter elements from yeast or fungi such as the Gal 4 promoter, theADH (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase)promoter, alkaline phosphatase promoter, and tissue-specifictranscriptional control regions derived from elastase I gene, insulingene, immunoglobulin gene, mouse mammary tumor virus, albumin gene,α-fetoprotein gene, α1-antitrypsin gene, β-globin gene, myelin basicprotein gene, myosin light chain-2 gene, and gonadotropic releasinghormone gene.

The nucleic acid constructs of the invention are inserted into anexpression vector or viral vector by methods known to the art, whereinthe nucleic acid molecule is operatively linked to an expression controlsequence. Also provided is a host-vector system for the production of atissue-specific fusion polypeptide of the invention, which comprises theexpression vector of the invention, which has been introduced into ahost cell suitable for expression of the fusion polypeptide. Thesuitable host cell may be a bacterial cell such as E. coli, a yeastcell, such as Pichia pastoris, an insect cell, such as Spodopterafrugiperda, or a mammalian cell, such as a COS, CHO, 293, BHK or NS0cell.

The invention further encompasses methods for producing the fusionpolypeptides of the invention by growing cells transformed with anexpression vector under conditions permitting production of thetissue-specific fusion polypeptides and recovery of the fusionpolypeptides so produced. Cells may also be transduced with arecombinant virus comprising the nucleic acid construct of theinvention.

The fusion polypeptides may be purified by any technique, which allowsfor the subsequent formation of a stable polypeptide. For example, andnot by way of limitation, the fusion polypeptides may be recovered fromcells either as soluble polypeptides or as inclusion bodies, from whichthey may be extracted quantitatively by 8M guanidinium hydrochloride anddialysis. In order to further purify the fusion polypeptides,conventional ion exchange chromatography, hydrophobic interactionchromatography, reverse phase chromatography or gel filtration may beused. The fusion polypeptides may also be recovered from conditionedmedia following secretion from eukaryotic or prokaryotic cells.

Therapeutic Methods

The invention herein further provides for the development of IGF fusionpolypeptides described herein as a therapeutic for the treatment ofpatients suffering from disorders which may be ameliorated by providingIGF1 or IGF2, for example, a condition caused or worsened by an IGFdeficiency. For example, a decrease in muscle mass, or atrophy, isassociated with various physiological and pathological states. Forexample, muscle atrophy can result from denervation due to nerve trauma;degenerative, metabolic or inflammatory neuropathy, e.g. Guillian-Barresyndrome; peripheral neuropathy; or nerve damage caused by environmentaltoxins or drugs. Muscle atrophy may also result from denervation due toa motor neuropathy including, for example, adult motor neuron disease,such as Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's disease);infantile and juvenile spinal muscular atrophies; and autoimmune motorneuropathy with multifocal conductor block. Muscle atrophy may alsoresult from chronic disease resulting from, for example, paralysis dueto stroke or spinal cord injury; skeletal immobilization due to trauma,such as, for example, fracture, ligament or tendon injury, sprain ordislocation; or prolonged bed rest. Metabolic stress or nutritionalinsufficiency, which may also result in muscle atrophy, include thecachexia of cancer and other chronic illnesses including AIDS, fastingor rhabdomyolysis, and endocrine disorders such as disorders of thethyroid gland and diabetes. Muscle atrophy may also be due to musculardystrophy syndromes such as Duchenne, Becker, myotonic,fascioscapulohumeral, Emery-Dreifuss, oculopharyngeal, scapulohumeral,limb girdle, and congenital types, as well as the dystrophy known asHereditary Distal Myopathy. Muscle atrophy may also be due to acongenital myopathy, such as benign congenital hypotonia, central coredisease, nemalene myopathy, and myotubular (centronuclear) myopathy.Muscle atrophy also occurs during the aging process. Muscle atrophy invarious pathological states is associated with enhanced proteolysis anddecreased production of muscle proteins.

The IGF1 and IGF2 fusion polypeptides of the invention are also usefulin diseases associated with an IGF deficiency, such as dwarfism. Stillfurther, IGFs have been shown to improve the survival of cardiac musclecells after an event such as a myocardial infarction, thus the fusionpolypeptides of the invention are useful in a subject who hasexperienced such an event.

The ability of the IGF fusion polypeptides of the invention to evade thelarge number of IGF-binding proteins present in a mammal makes themtherapeutically useful for efficiently treating conditions which maybenefit from an increased IGF level, such as recovery fromatrophy-promoting conditions, situations in which skeletal muscle masswas decreasing, or situations in which muscle hypertrophy is desirable,such as during recovery from immobilization, aging, cancer, etc.

Because IGF receptors are expressed broadly, IGF fusion molecules of theinvention wherein the fusion component is a multimerizing component suchas Fc or another active component could further be used in settingsother than muscle. For example, IGF1 and IGF2 have been shown to be bonegrowth factors, and therefore an IGF1-Fc or IGF2-Fc or an IGF1 or IGF2fusion to growth hormone could be useful in the treatment ofosteoporosis or other bone loss or weakness, including age relatedweakness, frailty or sarcopenia. The non-targeted molecules may also beuseful in settings of more general body mass wasting—such as cachexia.Cachexia is a condition causing body mass loss, including, but notlimited to, muscle mass. Settings of cachexia include cancer-inducedcachexia, AIDS-induced cachexia, sepsis-induced cachexia, renalfailure-induced cachexia, and congestive heart failure. Also, there isgrowth retardation in many settings, including thalassaemia, whichcauses short stature. Short stature in general would be a setting for anIGF1 or IGF2 fusion protein that is not targeted directly to muscle—suchas the IGF1-Fc or IGF2-Fc or the IGF1-GH or IGF2-GH embodiments. Anadditional use for IGF1 or IGF2 is to complement or substitute forinsulin. In settings of insulin-insensitive diabetes, IGF1 or IGF2fusion proteins of the invention may be used. Such variants may furtherbe used simply as a substitute for insulin in settings of hyperglycemia.Further additional uses for the IGF1 and IGF2 fusions described hereininclude use in the weaning of individuals from ventilators and for thetreatment of conditions such as anemia wherein the proliferation ofblood cells is desired.

Methods of Administration

Methods known in the art for the therapeutic delivery of agents such asproteins or nucleic acids can be used for the therapeutic delivery of anIGF fusion polypeptide or a nucleic acid encoding a IGF fusionpolypeptide of the invention, e.g., cellular transfection, gene therapy,direct administration with a delivery vehicle or pharmaceuticallyacceptable carrier, indirect delivery by providing recombinant cellscomprising a nucleic acid encoding an IGF fusion polypeptide of theinvention.

Various delivery systems are known and can be used to administer thefusion polypeptide of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987,J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part ofa retroviral or other vector, etc. Methods of introduction can beenteral or parenteral and include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, pulmonary,intranasal, intraocular, epidural, and oral routes. The compounds may beadministered by any convenient route, for example by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings(e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may beadministered together with other biologically active agents.Administration can be systemic or local. In addition, it may bedesirable to introduce the pharmaceutical compositions of the inventioninto the central nervous system by any suitable route, includingintraventricular and intrathecal injection; intraventricular injectionmay be facilitated by an intraventricular catheter, for example,attached to a reservoir, such as an Ommaya reservoir. Pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved, for example, and not by way oflimitation, by local infusion during surgery, topical application, e.g.,by injection, by means of a catheter, or by means of an implant, saidimplant being of a porous, non-porous, or gelatinous material, includingmembranes, such as sialastic membranes, fibers, or commercial skinsubstitutes.

In another embodiment, the active agent can be delivered in a vesicle,in particular a liposome (see Langer (1990) Science 249:1527-1533). Inyet another embodiment, the active agent can be delivered in acontrolled release system. In one embodiment, a pump may be used (seeLanger (1990) supra). In another embodiment, polymeric materials can beused (see Howard et al. (1989) J. Neurosurg. 71:105). In anotherembodiment where the active agent of the invention is a nucleic acidencoding a protein, the nucleic acid can be administered in vivo topromote expression of its encoded protein, by constructing it as part ofan appropriate nucleic acid expression vector and administering it sothat it becomes intracellular, e.g., by use of a retroviral vector (see,for example, U.S. Pat. No. 4,980,286), or by direct injection, or by useof microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

Cellular Transfection and Gene Therapy

The present invention encompasses the use of nucleic acids encoding thefusion polypeptides of the invention for transfection of cells in vitroand in vivo. These nucleic acids can be inserted into any of a number ofwell-known vectors for transfection of target cells and organisms. Thenucleic acids are transfected into cells ex vivo and in vivo, throughthe interaction of the vector and the target cell. The compositions areadministered (e.g., by injection into a muscle) to a subject in anamount sufficient to elicit a therapeutic response.

In another aspect, the invention provides a method of treating a targetsite, i.e., a target cell or tissue, in a human or other animalcomprising transfecting a cell with a nucleic acid encoding atissue-specific fusion polypeptide of the invention, wherein the nucleicacid comprises an inducible promoter operably linked to the nucleic acidencoding the targeting fusion polypeptide. For gene therapy proceduresin the treatment or prevention of human disease, see for example, VanBrunt (1998) Biotechnology 6:1149-1154.

Combination Therapies

In numerous embodiments, the fusion polypeptides of the presentinvention may be administered in combination with one or more additionalcompounds or therapies. For example, multiple fusion polypeptides can beco-administered in conjunction with one or more therapeutic compounds.The combination therapy may encompass simultaneous or alternatingadministration. In addition, the combination may encompass acute orchronic administration.

Pharmaceutical Compositions

The present invention also provides pharmaceutical compositionscomprising a fusion protein of the invention and a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the therapeutic is administered. Such pharmaceutical carriers canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Suitablepharmaceutical excipients include starch, glucose, lactose, sucrose,gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. The composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Where necessary, thecomposition may also include a solubilizing agent and a local anestheticsuch as lidocaine to ease pain at the site of the injection. Where thecomposition is to be administered by infusion, it can be dispensed withan infusion bottle containing sterile pharmaceutical grade water orsaline. Where the composition is administered by injection, an ampouleof sterile water for injection or saline can be provided so that theingredients may be mixed prior to administration.

The active agents of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freeamino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The amount of the fusion polypeptide of the invention which will beeffective in the treatment of a condition or disease can be determinedby standard clinical techniques based on the present description. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the condition, and should be decided according to thejudgment of the practitioner and each subject's circumstances. However,suitable dosage ranges for intravenous administration are generallyabout 20-5000 micrograms of active compound per kilogram body weight.Suitable dosage ranges for intranasal administration are generally about0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

Kits

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with at least one fusion polypeptide ornucleic acid encoding a fusion polypeptide of the invention. The kits ofthe invention may be used in any applicable method, including, forexample, diagnostically. Optionally associated with such container(s)can be a notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts, which notice reflects (a) approval by the agency ofmanufacture, use or sale for human administration, (b) directions foruse, or both.

Transgenic Animals

The invention includes transgenic non-human animals expressing an IGFfusion polypeptide of the invention. A transgenic animal can be producedby introducing nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. Any of theregulatory or other sequences useful in expression vectors can form partof the transgenic sequence. A tissue-specific regulatory sequence(s) canbe operably linked to the transgene to direct expression of thetransgene to particular cells. A transgenic non-human animal expressinga tissue-specific fusion polypeptide of the invention is useful in avariety of applications, including as a means of producing such fusionproteins. Further, the transgene may be placed under the control of aninducible promoter such that expression of the tissue-specific fusionpolypeptide may be controlled by, for example, administration of a smallmolecule.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 IGF Fusion Polypeptides are Cleaved at Lys 65 or 68

A fusion polypeptide was constructed utilizing human IGF1 containinghuman IGF1(Δ1-3, delR37) fused to human Fc. The polypeptide was purifiedand injected into mice. Blood samples were taken every 24 hours forseven days. Western analysis was conducted using an antibody specific tothe human Fc. After three days, a lower-migrating band was noticed,indicating that the IGF1-Fc was being proteolytically cleaved in serum.More serum was taken, and this lower-migrating species was purified andsequenced. It was established that the IGF1-Fc construct was cleaved atthe lysine 68 or the lysine 65 in the IGF1 variant. Based on thisresult, constructs are prepared in which the terminal three to six aminoacids are removed, or the lysine at position 65 or 68 is mutated toanother amino acid such as alanine or glycine. Such variants areutilized in preparation of fusion polypeptides of the inventioncomprising fusion components such as multimerizing components, targetingligands, and other active compounds, and are found to have greaterstability and half-life than comparable constructs with the lysine atposition 65 or 68 of the IGF1 variant component.

Example 2 IGF1-Fc Fusion Polypeptides

Fusion polypeptide constructs were made using IGF1variant-Fc, orFc-IGF1variant, utilizing the variant IGF1 (Δ1-3, delR37) and human IgGderived Fc. The activity of IGF1 was measured by its ability tophosphorylate the IGF1 receptor and Akt kinase. Such phosphorylation wasdetermined by a Western blot, using phospho-specific antibodies to thevarious molecules, or by immune-precipitating the receptors (such asIGFR), and Westerning with an antibody specific to anti-phosphotyrosine.Such blots indicated active phosphorylation of Akt using the constructIGF1-Fc, but little or no activity utilizing the construct Fc-IGF1.

IGF1(Δ1-3, delR37)-Fc was administered to SCID mice intra-peritoneallyor subcutaneously via daily injections or via injections every otherday. 4.5 mgs/kg of fusion protein were used for the injections. Controlmice got no IGF1-Fc protein. A third group of mice received both 15mg/kg dexamethasone subcutaneously per day. This dosage is sufficient toresult in 15% loss of muscle mass after twelve days. A fourth group ofmice received both the dexamethasone and the IGF1(Δ1-3, delR37)-Fc. Micein this fourth group experienced either no atrophy or statisticallysignificant reductions in atrophy (5% atrophy in tibialis anterior (TA)muscle for mice which received both dexamethasone and IGF1 (Δ1-3,delR37)-vs 15% atrophy of TA for those mice which received onlydexamethasone).

Example 3 IGF1-hGH Fusion Polypeptides

Fusion polypeptides were constructed utilizing IGF1(Δ1-3, delR37) withhGH as the fusion component. In addition to measuring IGF1 activity, theactivity of human growth hormone (hGH) was measured by its ability tophosphorylate Stat5. Surprisingly, constructs having the configurationIGF1variant-hGH caused phosphorylation of both IGF1 receptors and Stat5,while constructs with the configuration hGH-IGF1 variant had little orno activity.

Phosphorylation assays indicated that fusion proteins having theconfiguration IGF1 variant-hGH, which includes both hGH and the hIGF1variant described above, is capable of simultaneously activating theIGF1 Receptor, Akt, and Stat5. Such phosphorylation was determined by aWestern blot, using phospho-specific antibodies to the variousmolecules, or by immune-precipitating the receptors (such as IGFR), andWesterning with an antibody specific to anti-phospho-tyrosine. Inaddition, all of the above fusion polypeptides were made using humanIGF1 mutants which had the first three amino acids deleted (Δ1-3) andeither elimination or substitution of the arginines at positions 36and/or 37. Such mutant IGF1 molecules demonstrated both resistance tocleavage as well as reduced binding by IGF-1 binding proteins(specifically IGF1 binding protein 5) without affecting signalingability on C2C12 myotubes.

Fusion proteins comprising the IGF1 variants described above as well asdeletion of amino acids 65-70 or 68-70 or mutation of the lysine atposition 65 and 68 are constructed and tested for their ability tosimultaneously activate the IGF1 Receptor, Akt, and Stat5, as determinedby a Western blot, using phospho-specific antibodies to the variousmolecules, or by immune-precipitating the receptors (such as IGFR), andWesterning with an antibody specific to anti-phospho-tyrosine. Inaddition, C2C12 myotubes are contacted with the fusions and hypertrophymeasured as compared to hypertrophy caused by either IGF1 alone or hGHalone. In constructs in which a targeting ligand, such as agrin, is usedas a fusion component, activity may be measured by phosphorylation ofthe target receptor, such as MuSK (Glass et al. (1996) 85:513-523;Beguinot et al. (1988) Biochemistry. 27(9):3222-8).

Example 4 IGF2 Fusion Polypeptides

All of the above constructs are prepared utilizing the IGF2 protein orDNA as set forth in SEQ ID NOS:3-4. The lysine at position 65 of IGF2 isfound to be involved in the cleavage of fusion proteins comprising theIGF2 variant, supporting use of IGF2 variants with deletions in theterminal 3 amino acids (65-67).

Example 5 Effect of 2D-IGF1-Fc (Δ1-3, ΔArg37) on Blood Glucose

C57/BL6 mice were used at 13 weeks of age (n=3 mice per group). Micewere fasted for four hours. Baseline and post-injection blood sampleswere collected from the tail vein and blood glucose measured byglucometer. Either insulin, IGF1 or IGF1-Fc were administeredintraperitoneally and glucose was measured from blood collected from thetail vein 60 minutes after the administration of drug. Insulin wasadministered at 2 U/kg. IGF-I was used at a 10-fold higher dose thaninsulin (700 ug/Kg) based on comparative affinity of IGF-I for insulinreceptor. 2D-IGF1-Fc (hIGFΔ1-3, ΔArg37) was administered at doseequimolar to that of IGF-I (3.5 mg/kg).

Results: Insulin caused a 66% decrease in blood glucose (207±5.5 mg/dlbaseline vs. 70±22.2 mg/dl post-injection), IGF-I induced a 45% decreasein blood glucose (200±6.7 mg/dl baseline vs. 109±13.7 mg/dlpost-injection), and 2D-IGFI-Fc caused a 30% blood glucose decrease(184±7.3 mg/dl baseline vs. 129±15.9 mg/dl post-injection). Theseresults show that IGFI-Fc is effective in reducing blood gucose infasted C57BI/6 mice in an acute study.

Example 6 Pharmacokinetic Analysis of 2D-IGF1-Fc and 3D-IGF1-Fc

CD-1 mice (n=5 per group) were subcutaneously injected with 5 mg/kg ofeither human IGF1 derivative 2D-IGF1-Fc or 3D-IGF1-Fc (hIGF, Δ1-3,ΔArg37, Δ68-70). Serum samples were collected over a 7 day period andELISA assays performed with an anti-human IGF1 (USB Cat. No. 17661-05)and detecting antibody goat anti-human IgG.Fc-HRP (Jackson ImmunoResearch Cat. No. 109-035-098). The results are shown in FIG. 1.Pharmacokinetic parameters are shown in Table 1.

TABLE 1 Pharmacokinetic Parameters 2D-IGF1-Fc 3D-IGF1-Fc T_(MAX) hr 5.40 ± 1.12  6.00 ± 1.10 C_(MAX) mcg/mL  9.10 ± 1.75 13.27 ± 2.85T_(1/2) hr 24.09 ± 4.41 29.85 ± 5.51 AUC all mcg * hr/mL 325.42 ± 61.55 554.55 ± 112.23 AUCINF (obs) mcg * hr/mL 328.31 ± 62.10  566.82 ±114.63 V_(Z)(obs)/F mL/kg  535.77 ± 101.88 395.78 ± 82.00 CL(obs)/FmL/hr/kg 15.41 ± 2.91  9.26 ± 1.97 MRT last hr 32.69 ± 6.00 42.80 ± 7.87MRTINF (obs) hr 34.19 ± 6.27 42.46 ± 7.80

Example 7 IGFI-Fc Molecules Induce Skeletal Muscle Hypertrophy In Vivo

2D-IGFI-Fc and 3D-IGFI-Fc were injected subcutaneously into adult 8-weekold SCID mice daily at a dose of 1.6 mg/kg or with saline vehicle for 12days (N=5 per group). At the end of the experiment, muscles (tibialisanterior and the gastrocnemius complex) were removed and the muscle wetweight measured. Injection of 2D-IGFI-Fc caused a 23.07±2.14% (mean±SEM)increase in tibialis anterior mass and a 17.00±2.07% increase ingastrocnemius mass compared to control muscles. Injection of 3D-IGFI-Fccaused a 9.45±3.72% increase in tibialis anterior mass and a 9.31±2.15%increase in gastrocnemius mass compared to control muscles.

Example 8 2D-IGFI-Fc is Superior to IGF-I in Inducing Skeletal MuscleHypertrophy

The efficacy of 2D-IGFI-Fc in inducing skeletal muscle hypertrophy wasalso compared directly to unmodified IGF-I. Equimolar doses of each (4.8mg/kg of 2D-IGFI-Fc or 0.93 mg/kg of IGF-I) or saline vehicle wereinjected subcutaneously into adult 8-week old SCID mice every other dayfor 12 days (N=5 per group). At the end of the experiment, muscles(tibialis anterior and the gastrocnemius complex) were removed and themuscle wet weight measured. Injection of 2D-IGFI-Fc caused a 13.35%±2.26(mean±SEM) increase in tibialis anterior mass and a 9.39%±3.68% increasein gastrocnemius mass compared to control muscles. Injection of IGF-Igave 2.24%±2.27 increase in tibialis anterior mass and a 0.81%±1.51increase in gastrocnemius mass compared to control muscles.

1. A fusion polypeptide, comprising: (a) an insulin-like growth factor 1(IGF1) variant polypeptide component, comprising a human IGF1 protein ofSEQ ID NO:1 with the deletions of amino acid residues 1-3, 37, and65-70; and (b) a fusion component (F); wherein F is an Fc domain ofhuman IgG, wherein the IGF1 variant polypeptide component retains thefunctional activity of IGF-1.
 2. A dimer of the fusion polypeptide ofclaim
 1. 3. A nucleic acid encoding the fusion polypeptide of claim 1.4. A vector comprising the nucleic acid of claim
 3. 5. A host-vectorsystem comprising the vector of claim
 4. 6. The host-vector system ofclaim 5, wherein the host cell is selected from the group consisting ofa bacterial, yeast, insect, and mammalian cell.
 7. A method of producingthe fusion polypeptide of claim 1, comprising culturing a host celltransfected with the vector of claim 9, under conditions suitable forexpression of the polypeptide from the host cell, and recovering thepolypeptide so produced.
 8. A pharmaceutical composition comprising thefusion polypeptide of claim 1 and a pharmaceutically acceptable carrier.9. The fusion polypeptide of claim 1, wherein the IGF1 component and theFc domain are connected via a spacer sequence.